1. Introduction
Modified virus particles are used for multiple human medical applications such as vaccines. In such cases it is imperative that the virus composition used is purified such that potential medical risks, such as adverse side effects due to undesirable impurities, is eliminated, or as a minimum reduced to an acceptable level. Purification processes involve the removal of various impurities including unwanted residual DNA fragments originating from the cell substrate used to culture the virus. However, while the purification process must deliver products that meet the most stringent regulatory safety standards, the purification methods used must not degrade the medicinal properties of the virus itself, nor make the production process uneconomical so that the finished medical product becomes prohibitively expensive.
Purification techniques relying on the use of nuclease or chemicals to degrade the residual DNA into fragments too small to represent a significant clinical risk may result in degrading of the virus particles thereby detrimentally reducing the immunogenicity of the target virus. The various filtration, centrifuge and other techniques to clarifying the viral solution that rely on the differentiated physical properties of the desired virus as compared with the unwanted impurities (such as size weight, density and the like) may not adequately remove the DNA to levels that meet regulatory standards for medical products.
Purification techniques such as ionic chromatography and the like that rely on the differential electronic charges associated with the viral particles and the unwanted impurities may result in low production yields and thus high production costs.
The present invention provides a cost effective means to standardize the bulk production of highly purified viral compositions while retaining the immunogenicity of the virus particles. The present invention therefore meets the need to produce high quality, efficacious and affordable virus compositions that may meet international regulatory standards for safety and efficacy and are thus suitable for human medical applications. For over 45 years viruses for vaccines have been propagated using primary cells from various sources including; chicken embryos as well as monkey, dog, rabbit and hamster kidney cells. While products resulting from these manufacturing process are considered to be safe for human use, potential contamination by infectious pathogens, inconsistencies between individual animals and the ethical sensitivities of collecting animal cells resulted in the search for alternative cell substrates.
Diploid cells were found to overcome many of the shortcomings observed with primary cells. Diploid cells are well tolerated, non-tumorigenic, have a low frequency of chromosomal abnormalities and have a finite capacity for serial propagation. However, diploid cells are difficult to use in bulk quantities and environmental conditions are more stringent for successful use.
Continuous cell lines, a third class of cell substrate, are increasingly becoming the preferred option for virus production. The advantages of continuous cell lines are, the indefinite production of standard and well-characterized cells that grow efficiently and may be used for large-scale virus production in bioreactors. The WHO established a master cell bank for Vero cells, a continuous cell lines derived from the kidneys of African green monkeys being a cell line considered to be advantageous for the production of vaccine antigens (WHO Tech. Rep. 747 1987).
However, continuous cell lines themselves are associated with certain disadvantages relating to their use for medical purposes. Residual DNA may be capable of being integrated into the host genome, which may potentially result in malignant transformations of the host's/recipient's DNA leading to abnormalities in cell replication such as cancer.
Four factors impact the magnitude of the risk; the cell-substrate itself, different types have different safety profiles, the size of the DNA fragment i.e. below 200 base pairs is generally less than the size of a functional gene, the route of administration i.e. the up-take of DNA is approximately 10,000-fold less efficient in orally as opposed to parenterally administered compositions, and the concentration of DNA present.
The FDA's Code of Federal Regulations Title 21 part 610.13 under the general standards for biological products states that “Products shall be free of extraneous material except that which is unavoidable in the manufacturing process described in the approved biologics license application”. Specific regulations or guidelines have also been issued by the WHO and national regulatory bodies designed to control the level of DNA content in vaccine antigens derived from production processes using continuous cell lines.
The WHO expert committee on biological standardization technical report 878 issued in 1998 concluded that “The risk associated with residual continuous cell line DNA in a product is negligible when the amount of such DNA is 100 pg or less per parenteral dose.” The conclusion of the committee's assessment was that up to 10 ng of residual continuous-cell-line DNA per purified dose of vaccine was considered acceptable.
The European Agency for the Evaluation of Medicinal Products Committee for Proprietary Medicinal Products published a position statement in 2001 on the Use of Tumourigenic Cells of Human Origin for the Production of Biological and Biotechnological Medicinal Products cites the WHO conclusion above but also notes that the WHO report states that instances occur where the potential for clinical risks are higher, for example, where the residual DNA may contain infectious retroviral provirion sequences. The report concludes that the DNA level permitted in the final product “Should be as low as possible” and based on an assessment of the risk factors on a case-by-case basis.
The FDA's Guidance for Industry “Characterization and qualification of cell substrates and other biological materials used in the production of viral vaccines for infectious disease indications” issued in 2010 states that “You should limit residual DNA for continuous non-tumorigenic cells, such as low-passage Vero cells, to less than 10 ng/dose for parenteral inoculation as recommended by WHO.” The recommendation for orally administered compositions is less than 100 ug per dose given the naturally lower uptake for this administration method.
The 2010 Chinese Pharmacopeia issued by a committee headed by the Chairman of the Chinese Food and Drug Association (SFDA) states that the residual DNA from Vero cell substrates used in the production process for rabies vaccines must be less than 100 pg per dose i.e. 0.1 ng per dose or 100 fold less than the WHO recommendation (see above).
There is a continual medical need for a reliable large-scale supply of economically produced high quality virus compositions suitable for human medical applications including, but not limited to vaccines. In this context quality is defined as both, i) the antigenic properties of the virus such that it delivers the desired medical outcomes and ii) concurrently the purity of the viral compositions such that any contaminants introduced during the manufacturing process, for example DNA from the cellular substrate, are eliminated or reduced to an acceptable minimal level in the finished product to ensure that the composition is safe for human use.
One objective of the current invention is thus to propose a novel method for purifying viruses in very high yield which is easy to automate.
Another objective of the current invention is to propose a purification method that allows for whole, non-degraded viruses to be obtained.
A further objective of the current invention is to propose a purification method that significantly reduces concentration of unwanted impurities and specifically to reduce significantly the residual DNA from the cell line substrate in the finished product, thereby producing highly purified virus particles.
Yet another objective of the current invention is to propose a purification process that is scalable, i.e. it is possible to produce bulk quantities of the desired purified viral composition.
It is also an object of the present invention to improve the processing technology, simplify the equipment required, minimize the production costs, and ensure batch-to-batch consistency.
To achieve these aims, the subject of the invention is a method for purifying a viral culture, wherein according to the invention the purification of a viral culture is characterized by a method consisting of a combination of processes that includes at least one step to increase the ionic concentration of the viral composition and then at least one step to separate the desirable virus from the unwanted residual DNA by size exclusion chromatography. The result of this process is a composition of purified viruses that may be used to produce highly effective vaccines causing no significant adverse side effects in the recipients.
2. Description of Related Art
Methods for purifying viruses are known in the prior art. One approach to the purification of residual DNA is to degrade the DNA to a size small enough to mitigate the risk of functional residual fragments of DNA, that is, reducing the chances of the residual DNA retaining a functional gene to a clinically acceptable level.
One approach to the degradation of DNA is the introduction of nuclease, the enzyme selected on the basis of it being capable of breaking down the residual DNA fragments. Methods of using nuclease before or after cell lysation as described in the patent applications US 2009/0017523 and US2009/0123989. The degrading of the unwanted DNA may also be achieved by methods other than the introduction of nuclease, for example the use a chemical composition such as those described in patent application US 2009/0304729.
However, degrading the DNA fragments is not the preferable solution because the compositions, nuclease or otherwise, used to degrade the unwanted DNA tend also to have a detrimental effect on the immunogenicity of the target virus. To meet the overall objectives the resulting viral composition must have both a low residual DNA concentration and virus particles with the desired immunogenicity. Furthermore, the nuclease, chemical or other substance used to degrade the DNA must itself be then removed from the viral composition. Thus, in accordance with the current invention, the method of purification is characterized in part by the process of DNA removal or reduction without the use of nuclease.
Detergents may be use to precipitate DNA into a suspension within the viral composition thereby facilitating the removal of the DNA by means of filtration or centrifuge techniques. However, the detergent itself must then be removed from the final virus composition resulting in additional steps and costs in the manufacturing process. Thus, in accordance with the current invention, the method of purification is characterized in part by the process of DNA removal or reduction without the use of a detergent.
Size exclusion chromatography has been used to eliminate unwanted residual DNA from viral compositions, and specifically rabies virus compositions as produced on continuous cell line substrates, such as Vero cell (see Chtioui et. al. 2007, Kumar et. al. 2002 and Kumar et. al. 2005). Size exclusion, as described in the prior art, is conducted at low salt concentrations wherein “Low salt concentration” is exemplified by up to 0.5M NaCl. Such prior art publications fail to teach the benefits of using high ionic concentration buffers prior to the size exclusion chromatography to further enhance the removal of unwanted residual DNA.
Another approach described in the prior art is the use of ionic chromatography. Ionic chromatography has certain limitations in that the resulting yield of purified virus is significantly reduced, as compared with size exclusion chromatography (Purification of rabies virus produced on Vero cells using chromatography techniques, Chtioui et. al. 2007), making such ionic chromatography process unsuitable for large-scale virus production.
The advantage of the current invention is that while a significant reduction of residual DNA is achievable the overall process may be conducted at commercial scale with a high yield of virus suitable for medical use such as the production of vaccines.
3. Reference Information                U.S. 2009/0017523 Virus Purification Methods        U.S. 2009/0123989 Virus Purification Using Ultrafiltration        U.S. 2009/0304729 Cell-derived Viral Vaccines with Low Levels of Residual Cell DNA        U.S. Pat. No. 6,194,191 Method for the production and purification of adenoviral vectors        Purification of Rabies Virus Produced on Vero Cells Using Chromatography Techniques Chtioui et. al. Cell Technology for Cell Products, 629-634 2007        Process Standardization for Optimal Virus Recovery and Removal of Substrate DNA and Bovine Serum Proteins in Vero Cell-Derived Rabies Vaccine, Kumar et. al. Journal of Bioscience and Bioengineering, Vol. 94, No. 5, 3375-383, 2002        Purification, Potency and Immunogenicity Analysis of Vero Cell Culture-derived Rabies Vaccine: a Comparative Study of Single-step Column Chromatography and Zonal Centrifuge Purification, Microbes and Infection 7 (2005) 1110-1116 Kumar at. al. 2005        Chinese Pharmacopeia 2010        WHO Technical Report Ref. 747 issued in 1987        US FDA's Code of Federal Regulations Title 21 part 610.13        WHO Technical Report Ref 878 issued in 1998        European Agency for the Evaluation of Medicinal Products Committee for Proprietary Medicinal Products: position statement on the Use of Tumourigenic Cells of Human Origin for the Production of Biological and Biotechnological Medicinal Products issued in 2001        FDA's Guidance for Industry “Characterization and qualification of cell substrates and other biological materials used in the production of viral vaccines for infectious disease indications” issued in 2010        